Angiogenesis, the growth of new blood vessels, is essential for normal development, reproduction, and wound repair, but also contributes to tumor growth, atherosclerosis, and other diseases. The long-term goals of this project are a) to identify the key regulatory molecules responsible for new blood vessel growth, and b) to determine what stimuli induce their expression. The ovary is ideal for such studies because intense but self-limiting angiogenesis occurs normally during follicle and corpus luteum (CL) development. Granulosa cells, which play a central role in follicle and CL function, express vascular endothelial growth factor (VEGF), a potent endothelial cell mitogen. Expression of VEGF by granulosa cells in vitro is rapidly, strongly and specifically stimulated by hypoxia, the normal environment in the growing follicle and formative CL. The linkage of VEGF gene expression to hypoxia, a fundamental stimulus for angiogenesis, strongly suggests that VEGF is a physiological mediator of new blood vessel growth. The hypotheses to be tested in this proposal are 1) that hypoxia rapidly stimulates VEGF mRNA and protein production via hypoxia-responsive transduction molecules that in turn activate specific control sequences on the VEGF gene, and 2) that the VEGF subsequently regulates ovarian blood vessel growth and function. There are five Specific Aims: 1) To measure changes in VEGF expression in the rat ovary during periods of vascular remodelling, including a) the preovulatory period and CL formation, b) CL regressions, and c) placental assumption of CL support at mid-pregnancy using quantitative reverse transcription-polymerase chain reaction; 2) To determine the time course and dose response of the hypoxia-induce increases in VEGF mRNA expression in isolated granulosa cells; 3) To determine if the increased VEGF mRNA expression in accompanied by increased VEGF protein synthesis and secretion; 4) To determine whether hypoxia exerts a direct effect on VEGF gene expression at the transcriptional level and to identify the specific sequences on the VEGF gene required for hypoxic stimulation of VEGF expression; 5) To identify transcription factors that regulate VEGF expression in granulosa cells in response to hypoxia. These studies will significantly increase knowledge of the mechanisms by which hypoxia regulates the expression of VEGF and other genes that control physiological and pathological angiogenesis.